Liquid chromatography (LC) is a well-known technique for separating the constituent elements in a given sample. In a conventional LC system, a liquid solvent (referred to as the "mobile phase") is introduced from a reservoir and is pumped through the LC system. The mobile phase exits the pump under pressure. The mobile phase then travels via tubing to a sample injection valve. As the name suggests, the sample injection valve allows an operator to inject a sample into the LC system, where the sample will be carried along with the mobile phase.
In a conventional LC system, the sample and mobile phase pass through one or more filters and often a guard column before coming to the column. A typical column usually consists of a piece of steel tubing which has been packed with a "packing" material. The "packing" consists of the particulate material "packed" inside the column. It usually consists of silica- or polymer-based particles, which are often chemically bonded with a chemical functionality. When the sample is carried through the column (along with the mobile phase), the various components (solutes) in the sample migrate through the packing within the column at different rates (i.e., there is differential migration of the solutes). In other words, the various components in a sample will move through the column at different rates. Because of the different rates of movement, the components gradually separate as they move through the column. Differential migration is affected by factors such as the composition of the mobile phase, the composition of the stationary phase (i.e., the material with which the column is "packed"), and the temperature at which the separation takes place. Thus, such factors will influence the separation of the sample's various components. A more detailed description of the separation process can be found, among other places, in Chapters 2 and 5 of Introduction to Modern Liquid Chromatography (2d ed. 1979) by L. R. Snyder and J. J. Kirkland, which chapters are incorporated by reference herein.
Once the sample (with its components now separated) leaves the column, it flows with the mobile phase past a detector. The detector detects the presence of specific molecules or compounds. As discussed in Chapter 4 of Introduction to Modern Liquid Chromatography, which chapter is incorporated by reference herein, two general types of detectors are used in LC applications. One type measures a change in some overall physical property of the mobile phase and the sample (such as their refractive index). The other type measures only some property of the sample (such as the absorption of ultraviolet radiation). In essence, a typical detector in an LC system can measure and provide an output in terms of mass per unit of volume (such as grams per milliliter) or mass per unit of time (such as grams per second) of the sample's components. From such an output signal, a "chromatogram" can be provided; the chromatogram can then be used by an operator to determine the chemical components present in the sample.
In addition to the above components, an LC system will often include filters, check valves, a guard column, or the like in order to prevent contamination of the sample or damage to the LC system. For example, an inlet solvent filter may be used to filter out particles from the solvent (or mobile phase) before it reaches the pump. A guard column is often placed before the analytical or preparative column; i.e., the primary column. The purpose of such a guard column is to "guard" the primary column by absorbing unwanted sample components that might otherwise bind irreversibly to the analytical or preparative column.
It will be understood to those skilled in the art that, as used herein, the term "LC system" is intended in its broad sense to include all apparatus used in connection with liquid chromatography, whether made of only a few simple components or made of numerous, sophisticated components which are computer controlled or the like.
Many different types of LC systems and components for LC systems are commercially available from a number of vendors. For example, Millipore Corporation of Milford, Mass., Beckman Instruments of Fullerton, Calif., and Hewlett-Packard Co. of Palo Alto, Calif., all sell LC systems, including pumps, sample injection valves, columns, and detectors, among other things. In addition, various columns with various packings are commercially available from a variety of sources, including (among others) Upchurch Scientific, Inc., of Oak Harbor, Wash., and Baxter Healthcare Corporation of Deerfield, Ill.
Today, most LC systems include pumps which can generate relatively high pressures of up to around 6,000 psi. In many situations, an operator can obtain successful results by operating an LC system at "low" pressures of anywhere from just a few psi or so up to 1,000 psi or so. More often than not, however, an operator will find it desirable to operate an LC system at relatively "higher" pressures of over 1,000 psi. The operation and use of LC systems at such "higher" pressure levels is often referred to as "high pressure liquid chromatography" or "high performance liquid chromatography" (HPLC).
In order to be suitable for HPLC applications, a column must be made to withstand the typical operating pressures of the LC system. If the column is too weak, it may burst and thereby leak. Given the types of solvents that are sometimes used as the mobile phase and the expense of obtaining and/or preparing many samples for use, any such failure is a serious concern. Besides being able to withstand such pressures, the column must be made of a material which can withstand the chemical action of the mobile phase; i.e., the ideal column is chemically inert to the mobile phase used. In addition, the column needs to be durable so that it has a commercially useful life span.
Given such concerns, conventional columns typically consist of a stainless steel tube which had stainless steel end fittings attached at each end. Often, such columns were "packed" with appropriate materials to achieve the chemical separation required. A detailed discussion of end fittings, and column end fittings in particular, can be found in Chapter VII of the booklet HPLC Fittings (2d ed. 1992) by Paul Upchurch, which chapter is incorporated by reference herein. Typical column end fittings must hold to operating pressures of up to 6,000 psi or so. Such conventional column end fittings usually have been machined from stainless steel. The end fittings also must be suitable for connecting the column to the tubing used in the LC system to connect the various elements of the LC system with one another.
The inside diameter of the column must be polished in order to eliminate the possible adverse effects a rough inside wall may have on the separation process. It has been suggested that the smoothness of the inside wall of the column influences the homogeneity of the packing. Hence, a smoother surface finish on the column's inside surface results in a better level of performance from the column. Accordingly, most conventional columns typically consist of a stainless steel tube with a highly polished inside diameter surface finish. Although such a polished finish is important, it requires additional manufacturing steps and can be expensive to obtain.
More recently, it has been realized that the use of stainless steel components in an LC system have potential drawbacks in situations involving biological samples. For example, the components in a sample may attach themselves to the wall of a stainless steel column. This presents problems because the detector's measurements (and thus the chromatogram) of a given sample may not accurately reflect the sample if some of the sample's ions remain in the column and do not pass the detector. Perhaps of even greater concern, however, is the fact that ions from the stainless steel column may detach from the column and flow past the detector, thus leading to potentially erroneous results. Hence, there is a need for a "biocompatible" column; i.e., a column made of a material which is chemically inert with respect to such "biological" samples and the mobile phase used with such samples so that ions will not be released by the column and thus contaminate the sample. It will be understood that the use of the term "column" herein applies to analytical and preparative columns, and also applies to guard columns and the like.
To avoid biocompatibility problems, glass lined columns which have an exterior made of stainless steel are known. Because such columns are prone to breakage, a great deal of care in the manufacture, transportation, handling, and use of such columns is required. Moreover, it has been observed that the use of such glass-lined columns at high pressures is not appropriate.
Columns using extruded rods or tubing made of the polymer polyetheretherketone (PEEK) with molded or machined PEEK column end fittings are also known, but because of their lack of strength, such columns are typically limited to applications using fairly low pressures (i.e., pressures of not more than 200 psi or so), although some columns with larger walls have been claimed to work at up to 2,000 psi or so. Columns made of the polymer PEEK are also known, but such columns are machined from large diameter PEEK rods. In order to produce such PEEK columns in this way, the PEEK tube must first be machined and then its inside diameter must be polished. This approach thus involves additional manufacturing steps which add additional costs. Also, the surface finish obtained in such columns, while acceptable for many LC applications, is often unacceptable for applications where high levels of performance are required of the column. Moreover, to hold to the high pressures at which many columns are packed and at which some LC systems operate, such PEEK columns must be made with a very thick wall.
Another conventional column includes a PEEK tube surrounded by an aluminum jacket. However, the use of PEEK tubes still requires the extra manufacturing costs and therefore added time and expense. In addition, the surface finish obtained via such an approach may not be acceptable for LC applications requiring high performance by the column. Such columns also require the use of specialized, metallic end fittings which can handle the higher pressures but which require the use of additional polymeric pieces to ensure biocompatibility. The use of such additional pieces results in a higher chance that a connection between some of the pieces will leak or fail, an obviously undesirable result. The additional pieces and joints in such columns also may decrease the durability of such columns.
Therefore, it is an object of the present invention to provide a biocompatible column which can be used in relatively high pressure LC applications and which has sufficient strength for use in relatively high pressure LC applications.
It is yet another object of the present invention to provide a biocompatible column which is not fragile and which does not require excessive manufacturing steps or costs.
It is still another object of the present invention to provide a biocompatible column which is easily connected or disconnected.
It is still another object of the present invention to provide a biocompatible column with a highly polished inside surface and with a minimal number of pieces.
It is still another object of the present invention to provide a durable, biocompatible column which can provide a high level of performance at relatively high pressures with a minimal chance of leakage.
The above and other advantages of the present invention will become readily apparent to those skilled in the art from the following detailed description of the present invention, and from the attached drawings, which are briefly described below.